Method for detecting Cryptosporidium parvum oocysts

ABSTRACT

Methods for detecting parasites, such as  Cryptosporidium parvum , in turbid and non-turbid samples by solubilizing molecular markers or antigens of the parasite. The molecular markers are solubilized by incubating a sample containing the parasite with a solubilization buffer and detecting the solubilized antigens by electrochemiluminescence. The solubilization buffer contains one or more detergents alone or in combination with one or more denaturing agents in a buffered solution. The methods are an improvement over existing immunofluorescence assays for  C. parvum  because the methods described herein are quantitative, reproducible, have high sensitivity, are not labor-intensive, require only minimal sample processing, and avoid being adversely affected by sample turbidity. In addition, by using a electrochemiluminescence assay, microscopy is not required.

This invention was made in the Centers for Disease Control. Therefore,the United States Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the field of detection assays and moreparticularly to an improved method for detecting Cryptosporidium parvumoocysts.

BACKGROUND OF THE INVENTION

Parasitic infections of the gastrointestinal tract are prevalent aroundthe world. Many gastrointestinal parasites are transmitted by theconsumption of contaminated food or water. Although gastrointestinalparasitic infections in the general population cause abdominal disordersfor only a short period of time, in the immunocompromised individual, aparasitic infection can be deadly.

Cryptosporidium parvum (C. parvum) is a food or waterborne parasite thatinfects humans and animals causing severe intestinal distress. Since the1970's, C. parvum has been receiving increased world wide attention. Inthe early 1980's following two outbreaks of C. parvum infections in theUnited Kingdom resulting in a total of 516 cases, the British governmentwas compelled to devise a method for detecting C. parvum in water. (K.M. Shepherd et al., APPLD. AND ENVIRON. MICRO., Vol. 62, No. 4 pp.1317-1322 (1996)). In the United States, waterborne outbreaks of C.parvum are being reported with increasing frequency. One of the latestoutbreaks took place in Milwaukee, Wis. in April 1993 involving theinfection of an estimated 400,000 people. (C. Drozd et al. APPLD. ANDENVIRON. MICRO., Vol. 62, No. 4 pp. 1227-1232 (1996)). Infection causedby C. parvum is particularly dangerous because it can cause prolongeddiarrheal illness that may be potentially fatal for immunocompromisedindividuals.

C. parvum is a parasite that infects its host by invading the intestinaland urogenital systems. C. parvum organisms may be transmitted in avariety of ways including via contaminated food or water, animal toanimal contact, via farm animals such as sheep and calves, oralternatively by oocysts in feces. Human infections generally resultfrom zoonotic spread, person-to-person contact, fecal-oral contact,oral-anal contact or waterborne transmission. Although,cryptosporidiosis occurs worldwide, children, travelers to foreigncountries, male homosexuals, and medical personnel caring for patientswith the disease, are at particular risk. In developed countries, 1 to4% of children with gastroenteritis harbor C. parvum oocysts; and indeveloping countries, 4 to 11% of such children have cryptosporidiosis.Apart from humans, Cryptosporidium infections are widespread in severalother vertebrates such as mammals, reptiles and fish: and accordingly,the frequency of cryptosporidiosis is reported to be relatively high foranimal handlers and veterinarian personnel.

Unlike other coccidia, C. parvum is found on the brush border ofintestinal epithelium and not within deep intracellular regions.Typically, C. parvum organisms are small (2 to 6 μm) spherules thatinhabit the microvillus border of the intestinal epithelium arranged inrows along the brush border of the jejunum. After introduction into theintestine, C. parvum sporozoites attach to the microvilli surfaces andreproduce by schizogony (asexually). The resulting infective oocysts arepassed into the intestinal lumen and passed in the feces. Followingingestion of the oocysts by another vertebrate, the oocysts releasesporozoites that attach themselves to the epithelial surface andinitiate a new cycle of infection.

As C. parvum organisms invade the surface of intestinal cells, the hostexperiences symptoms such as reduced appetite, severe diarrhea andchronic fluid loss. In normal hosts, the onset of the disease isexplosive, with profuse, watery diarrhea and abdominal cramping thatlasts from 4 to 14 days following exposure. The symptoms generallypersist for 5 to 11 days, and then rapidly abate as remission of theparasite occurs in about 10-15 days. However, in immunocompromisedindividuals, (i.e. marasmic and malnourished children, individuals withcongenital hypogammaglobulinemia, those receiving immunosuppressants forcancer therapy or organ transplantation, and patients with AIDS), onsetof the disease is more gradual and diarrhea is more severe, with dailyfluid losses of up to 15 to 20 liters. Unless the underlying immunologicdefect is corrected, the diarrhea may continue persistently orremittently for life. (Merck Manual, Chapter 15 p. 237 16th ed. (1992)).

There is no effective, specific anti-C. parvum therapy available atpresent. Although some patients have responded positively to therapywith conventional antibiotics such as spiramycin and paromomycin, theresult of infection is frequently fatal for immunocompromisedindividuals. In fact, cryptosporidiosis is one of the predominant causesof death in immunocompromised patients.

In light of the potential disastrous consequences of C. parvuminfection, sensitive, efficient methods for detecting C. parvumcontamination are necessary. In humans, the typical source ofcryptosporidiosis is contaminated water, therefore safeguarding watersupplies is a primary goal. The United States Environmental ProtectionAgency has recognized the necessity for improved detection methods byinitiating the establishment of mandatory guidelines for C. parvumlevels in drinking water.

Currently available detection systems indicate that C. parvum organismsare observed in “spikes”; meaning that levels of C. parvum in samplescollected upstream and downstream, from the same source of thecontamination, may not be identical when simultaneous readings are made.Consequently, C. parvum levels recorded from one location may differsignificantly from readings taken from the same location minutes later.Detection of C. parvum in water is further complicated because theinitial source of infection is difficult to identify. An abnormally highC. parvum concentration may be caused by water run-off from contaminatedfarm or pasture land, or an infant's soiled diaper carelessly discardedinto a stream.

Ideally, continuous filtration systems having the capability to captureand retain C. parvum organisms for subsequent analysis would beinstalled in all water supply reservoirs to allow for continuousmonitoring. Unfortunately, filtration systems currently in use oftenhave filtration cartridges that either fail to retain organisms,frequently become clogged with mud or sediment, or must be replaced orcleaned with a frequency that renders the cartridges impractical.

C. parvum detection assays presently in use are cumbersome andfrequently inaccurate. For example, most assay test samples begin ascrude mixtures of C. parvum oocysts separated out from mud depositscollected by filters. The oocysts are isolated by processes involvingcentrifugation and ultrafiltration. Separating oocysts in this manner isoften tedious and inefficient since each time the test sample is spunand filtered, oocysts are lost in the process, inevitably resulting inlack of sensitivity and related inaccuracies. Another significantdisadvantage of such assays is the large amount of time required forprocessing test samples. For example, in order to improve the opticalproperties of test samples for detection, oocysts must be stained.Typically, staining and subsequent detection procedures can take up tofour days. Furthermore, samples can be tested only in small increments(i.e. 50 μl), and the sensitivity of most currently available assays isvery low. Generally at least 50,000 C. parvum oocysts per millilitermust be present for a positive detection result. Therefore, C. parvumassays currently in use are generally inefficient, inaccurate andinconsistent.

Another barrier to effective Cryptosporidium screening concerns sampleturbidity. The term “turbidity” refers specifically to the clarity ortransparency of water and the effect that any suspended particles in thewater may have on this clarity. Turbidity is determined by quantifyingthe amount of light allowed to pass through a sample and is measured inNTUs (nephelometric turbidity units). Many source water sites of publicwater reservoirs, e.g. rivers and lakes, often have turbidities up to100 NTU, whereas finished water, e.g. reservoirs for public consumption,tend to have turbidities in the range of 0 to 5 NTU.

Because it is commonly suspected that Cryptosporidium contaminationoccurs at source water sites, efforts have been focused on assayingsamples at reservoir intakes. Several gallons of source water are pumpedthrough filters that are rated to capture particles the size of oocystsor larger. Pumping source water in this way causes large amounts ofsediment to obstruct the flow of water through filters and thereforelimit the volume of water passing through the filters. The filterretentates are then eluted and assayed for the presence ofmicroorganisms. These retentates can have turbidities up to 300,000 NTUand yield highly variable C. parvum oocyst counts by immunofluorescenceassay due to the loss of oocysts that occurs in multi-step sampleprocessing.

Oocysts present in filter eluate often tend to be washed away duringprocessing and therefore go undetected in the final step of detectionassays. Consequently, currently available methods such asimmunofluorescence assays (IFA) and enzyme immunoassays (EIA), aremainly useful for detecting oocysts in “clean” samples, i.e. samplesthat have low turbidity. Such assays are more likely to givereproducible results with clean samples than those that are considered“dirty”, i.e. samples that have high turbidity.

Clinical diagnosis of cryptosporidiosis is made by recovering acid-fastoocysts from stool samples. Excretion of acid-fast oocysts is mostintense during the first four days of illness but persists for theduration of diarrhea. Other assays currently in use for diagnosticpurposes involve the use of formalin-ethyl acetate sedimentation orSheather's sugar flotation stool concentration procedure to enhance theyield of oocysts in specimens containing few oocysts. Commercialfluorescein-labeled monoclonal antibody kits also provide detection ofoocysts in clinical specimens. (Merck Manual, Chapter 15 p. 237 16th ed.(1992)). The disadvantage of such clinical tests is that depending onthe stage of C. parvum infection, the assays may or may not beadequately sensitive for detecting oocysts. In addition, such clinicaltests generally involve a multitude of steps thereby introducing agreater likelihood of inaccuracies. Furthermore, no “standard” fortesting stool specimens for C. parvum has been established, and so theabsolute sensitivity of currently used methods has not been assessed.(Christine L. Roberts et al., JOURN. OF CLIN. MICRO., Vol. 34 No. 9, pp.2292-2293 (1996)). Other problems associated with C. parvum testinginclude extensive processing time and low test positivity rates.

In summary, existing assays for parasites such as C. parvum areirreproducible, insensitive, labor-intensive, susceptible tointerference by sample turbidity, and time consuming. In addition,existing assays are not quantitative, and scientific data correlatingparasite levels in drinking water with incidence and severity of diseasein healthy and immunocompromised persons, are unavailable. Useful assaysthat enable correlation of disease-parasite levels are required for thedevelopment of environmental guidelines for safeguarding water sourcesagainst C. parvum and other parasitic infestation. What is neededtherefore, is a sensitive, quantitative and reproducible assay for foodor waterborne parasites that can efficiently process numerous sampleswithout requiring a multitude of steps and subjective determinations.

SUMMARY OF THE INVENTION

An efficient and sensitive method for the detection of food orwaterborne parasites, such as C. parvum oocysts, is provided. Inaccordance with the method, molecular markers of the parasite aresolubilized, thereby allowing recognition and detection of the parasitein turbid samples. Unlike prior art assays, the results of the assaydescribed herein are both reproducible and quantitative. In addition theassay is especially useful for detecting C. parvum oocysts present insource water such as recreational water and natural bodies of water,finished water such as community water reservoirs, biological fluidsamples, or fecal samples, without interference from sample turbidity.The assay is highly sensitive, allowing for the detection of less than50,000 oocysts per milliliter, and permits rapid sample testing.

The assay described herein includes the steps of solubilizing molecularmarkers, or antigens, of the parasite and detecting the antigens byimmunological means. Preferably the assay is useful for detecting C.parvum in test samples by solubilizing oocyst antigens and detecting thesolubilized antigen, preferably by magnetic matrix capture andelectrochemiluminescence. The assay is particularly suited for parasitedetection in environmental water, biological fluid samples, and fecalsamples because it can operate in both low and high turbidity samples,has high reproducibility, and can be performed with little samplemanipulation or processing.

Accordingly, it is an object of the present invention to provide aqualitative or quantitative assay for the detection of food orwaterborne parasites such as C. parvum oocysts.

It is another object of the present invention to provide a detectionassay for parasites, such as C. parvum oocysts, in either source orfinished water.

It is yet another object of the present invention to provide a detectionassay for parasites, such as C. parvum oocysts, in turbid samples,particularly turbid water samples.

It is another object of the present invention to provide a detectionassay for food or waterborne parasites, such as C. parvum oocysts, inbiological samples such as biological fluid samples and stool, or fecalsamples.

Another object of the present invention is to provide a quantitativedetection assay enabling the correlation of parasite levels in the foodor water source, such as C. parvum oocyst levels in drinking water, andthe incidence of disease in normal, healthy individuals, orimmunocompromised individuals.

Yet another object of the present invention is to provide a method forsolubilizing molecular markers of parasites, such as C. parvum oocysts.

Another object of the present invention is to provide a kit for anoptimized assay configuration for automated point-of-use analysis fordetecting parasites, such as C. parvum, in water, biological fluids, orfecal samples.

Another object of the present invention is to provide a method for theimmunological detection of parasites, such as C. parvum oocysts, thatutilizes electrochemiluminescence technology.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing titration curves, measured byelectrochemiluminescent counts, of C. parvum oocysts in buffer orsediment samples having various nephelometric turbidity unit (NTU)values.

FIG. 2 is a bar graph showing a comparison of oocyst detection, measuredas electrochemiluminescent counts per sample, in buffer versus sedimentsamples. The graph demonstrates that sediment samples with up to 330,000NTU can be assayed using the method described herein for the presence ofOW3 antigens in spiked samples.

FIG. 3 is a bar graph showing electrochemiluminescent counts for ninedifferent parasites, measured using the detection assay described hereinwith a monoclonal antibody specific for C. parvum.

FIG. 4 is a bar graph of differential electrochemiluminescent counts forC. parvum and a related species, C. baileyi, demonstrating that thedetection assay described herein is specific for C. parvum and does notcross-react with Cryptosporidium baileyi.

DETAILED DESCRIPTION

A method or assay for the detection of food or waterborne parasites isprovided. The method is useful for the detection of parasites in avariety of samples including, but not limited to, source water such asrecreational water and natural bodies of water, finished water such ascommunity water reservoirs, biological fluid samples, fecal samples, andother turbid samples without interference from sample turbidity. Theassay permits rapid sample testing and is highly sensitive, allowing thedetection of less than 50,000 oocysts of the parasite per milliliter.Preferably, the assay is capable of detecting oocysts at a concentrationas low as 10,000 oocysts per milliliter, most preferably as low as 1,000oocysts per milliliter.

In accordance with the method provided herein, molecular markers of theparasite are solubilized and the solubilized molecular markers aredetected with an immunologic assay, preferably utilizingelectrochemiluminescence. The method is particularly advantageousbecause microscopy is not required and the results lack susceptibilityto influence by turbidity. In addition, the results are bothquantitative and reproducible.

The term “molecular marker” is defined herein as an antigen of theparasite. Preferably, the antigen is a membrane-bound protein orglycoprotein. Most preferably the antigen is unique to the parasitebeing detected by the assay and is found on the surface of an oocyst ofthe parasite.

Preferably, the assay detects parasites such as Plasmodium (particularlyPlasmodium falciparum), Trypansoma (particularly Trypansoma cruzi),Cyclospora, Giardia, and Cryptosporidium. More preferably, the assaydetects Cryptosporidium parvum (C. parvum). Most preferably, the assaydetects solubilized antigens or molecular markers of C. parvum oocysts.

Molecular Marker Solubilization

One or more molecular markers of the parasite to be detected in asample, such as membrane-bound proteins or glycoproteins of the oocyststage of the C. parvum organism, are solubilized by combining a samplecontaining the parasite with a solubilization buffer for a sufficientamount of time and under conditions that facilitate proteinsolubilization without resulting in protein denaturation to the extentthat it would impair the formation of an antibody-antigen complexbetween the solubilized molecular marker and an antibody havingspecificity for the molecular marker.

The solubilization buffer is a buffered solution containing one or moredetergents alone or in combination with one or more denaturing agents.Preferably, the solubilization buffer for C. parvum oocyst antigenscontains a detergent in a mildly alkaline buffer solution.

In general, membrane-bound proteins or glycoproteins present on thesurface or outer wall of the parasite, preferably the oocyst stage ofthe parasite, are solubilized using a wide range of detergents,denaturing agents, or a combination of detergents and denaturing agents,in a buffered solution. Exemplary detergents and denaturing agentsinclude, but are not limited to, Zwittergent™ detergent, Tween-20™detergent, Triton X-100™ detergent, NP40™ detergent, urea, sodiumdodecyl sulfate, guanidine hydrochloride. The preferred pH is mildlyacidic or mildly alkaline. The term mildly acidic is defined herein asless than pH 7 and greater than pH 4. The term mildly alkaline isdefined herein as greater than pH 7 and less than pH 10. The optimalformulation of solubilization buffer depends on the physicochemicalproperties of the molecular marker to be solubilized and detected by thedetection assay.

For example, in a preferred assay, the antigen to be detected is thetarget epitope of OW3, which is found on the C. parvum oocyst cell walland is currently the desired molecular marker for C. parvum detection insome of the available immunofluorescence assays (IFA). In a preferredembodiment of the detection method described herein, OW3 antigens aresolubilized with a solubilization buffer containing Zwittergent™detergent and Tris-HCl, at alkaline pH, at a temperature above roomtemperature for at least 30 minutes. More preferably, C. parvum OW3antigens are solubilized with a solubilization buffer containing lessthan 1% Zwittergent™ 3-10 detergent and less than 0.1 M Tris-HCl (pH8-10) at a temperature greater than 75° C. for between 30 and 90minutes. Most preferably, C. parvum OW3 antigens or epitopes aresolubilized with a solubilization buffer containing approximately 0.65%Zwittergent™ 3-10 and approximately 0.05M Tris-HCl, approximately pH8.0, at approximately 95° C., for approximately one hour.

The solubilization of the molecular markers, or surface antigens, ofother parasites or oocysts of parasites, can be determined by thoseskilled in the art by spiking a sample with a known amount of parasiteto be detected; adjusting the choice and concentration of detergent anddenaturing agent, pH, temperature, length of incubation, and degree ofagitation; and maximizing the conditions that yield the highestconcentration of solubilized, or detectable, antigen by immunoassay.

Antigen Capture and Detection

Following solubilization, the solubilized marker or markers are reactedwith one or more antibodies that bind to the solubilized antigen underconditions that facilitate antibody-antigen complex formation. Theantibodies may be monoclonal or polyclonal antibodies. Most preferably,the antibodies are monoclonal antibodies specific for the solubilizedantigen. The antibodies are prepared in accordance with methods known tothose skilled in the art. The molecular markers function asrepresentatives of the organism being detected by the assay.

For example, epitopes of OW3 are representative of C. parvum. OW3 isparticularly selected because the epitopes of OW3 are present inabundance on the C. parvum oocyst wall, but not on other Cryptosporidiumspecies or other parasites.

Preferably, the antibody used to capture the molecular marker is amonoclonal antibody or antibodies bound to a solid phase. Exemplarysolid phase substances include, but are not limited to, microtiterplates, test tubes, magnetic, plastic or glass beads and slides.Preferably, magnetic beads coated with monoclonal antibodies (MAb)specific for epitopes of membrane-bound protein antigens are used tocapture the soluble antigens.

The antibody-antigen complexes are then detected using immunoassaymethods known to those skilled in the art, including sandwichimmunoassays and competitive immunoassays. The antibody-antigencomplexes are exposed to the same monoclonal antibodies as those used tocapture the antigen, but which have been labeled with a detectablelabel. Suitable labels include: chemiluminescent labels, such ashorseradish peroxidase; electrochemiluminescent labels, such asruthenium and aequorin; bioluminescent labels, such as luciferase;fluorescent labels such as FITC; and enzymatic labels such as alkalinephosphatase, β-galactosidase, and horseradish peroxidase. Preferably,the label is detected by electrochemiluminescence. Most preferably, thedetecting monoclonal antibody is modified by the addition of a ruthenium(Ru²⁺) label.

The labeled complex is then detected using a detection technique orinstrument specific for detection of the label employed. Preferably, thecomplexes are analyzed by an electrochemiluminescence instrument such asthe ORIGEN™ ANALYZER (Igen, Inc., Gaithersburg, Md.) for Ru²⁺ photonemission. Soluble antigen or antigens may also be incubated withmagnetic beads coated with non-specific antibodies in an identical assayformat to determine the background values of samples analyzed in thepresent assay.

In a preferred embodiment of the present method, solubilized antigen iscomplexed with monoclonal antibody-coated magnetic beads and, the beadsare captured by an electromagnet present in the flow cell of the ORIGEN™ANALYZER. After capture, the Ru²⁺ present in immuno-complexes istriggered to release a photon of light by the addition of the electrondonor tripropylamine followed by the application of an electrical field.Light measured by a photomultiplier tube is then expressed inelectrochemiluminescent (ECL) counts. Samples positive for the parasite,such as C. parvum, will yield ECL counts above a set background signal.

Assay Characteristics

The detection assay described herein is efficient because of the rapidrate at which samples may be screened, processed and analyzed.Typically, samples of approximately 1 ml may be analyzed at a rate ofabout 60 seconds per sample. In addition, the assays of the presentinvention have improved efficiency due to the higher recovery ofparasites as a result of limited sample processing. The detection methodis a rapid and quantitative assay that is not disrupted by test sampleturbidity. The assay time of the present invention for detecting C.parvum oocysts in source or finished water and in fecal sample isapproximately six hours, while the existing microscopic assays for thisorganism in water samples can take up to four days to complete.

The method described herein can successfully detect C. parvum oocysts inone milliliter samples containing less than 50,000 oocysts, and canpreferably detect as few as 10,000 oocysts/ml. Most preferably, themethod can successfully detect 1,000 oocysts/ml or less. This is asignificant improvement over the sensitivity of presently availablemethods that fail to detect C. parvum in one milliliter samplescontaining less than 50,000 oocysts.

As shown in FIGS. 1 and 2, the detection assay of the present inventionis not subject to interference by sample turbidities and can properlyprocess samples having up to 330,000 NTU. In comparison, currentmethodologies such as immunofluorescence assays (IFA) and enzymeimmunoassays (EIA) are time consuming and severely limited by sampleturbidity. Since sample turbidity is not a major impairment tosuccessful detection by the method described herein, the assay issuitable for detecting oocysts in samples of source or finished waterfiltrate obtained from the eluate of filter cartridges (which commonlyexhibit high turbidities as high as approximately 300,000 NTU).Furthermore, as demonstrated by FIGS. 3 and 4, the detection assay ofthe present invention is specific for C. parvum: the assay does notcross-react with other species of Cryptosporidium.

The assay is also valuable for epidemiological reasons as it may be usedto identify low-level infections in patients. This is especiallyimportant because existing assays for C. parvum have low sensitivitymaking the detection of asymptomatic cryptosporidiosis a formidabletask. Unlike the assay described herein, presently available assays aregenerally considered inaccurate and inefficient due to the variation inconsistency between individual samples, the variation in amount ofspecimen used, and oocyst losses incurred during laborious samplepreparation.

Unlike assays currently used in the art, the presently described methoddetects the organism by recognition of the molecular markers of theorganism. The advantage of this type of recognition is that the assay isneither dependent upon recognizing the parasite in particulate form orupon detecting the presence of oocysts that are intact. Instead theassay is directed at detecting the presence of solubilized antigens thatare present in abundance on oocyst cell walls. Detection based on thepresence of solubilized cell wall molecular markers both increases thesensitivity of the claimed detection method, and reduces interferenceresulting from sample turbidity.

The invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention.

EXAMPLE 1 Preparation of Solubilized C. Parvum Oocyst Antigens

C. parvum oocyst membrane-bound antigens present on the oocyst cellwall, more specifically epitopes of OW3 antigens, were solubilized inaccordance with the following procedure.

Aliquots containing 500 μl of test samples, negative and positivecontrols containing known number of C. parvum oocysts (provided by Dr.M. J. Arrowood, CDC, Atlanta, Ga.), were placed in 1.7 ml siliconizedmicrotubes and microfuged at 21,000×g for five minutes at 4° C. Thesupernatants were discarded and the pellets resuspended in 500 μl ofsolubilization buffer and incubated for 60 minutes.

When utilizing the OW3 monoclonal antibody for detecting C. parvumoocysts, a solubilization buffer containing 0.65% by weight Zwittergent™3-10 detergent (Calbiochem-Novabiochem, La Jolla, Calif.), 0.05MTris-HCl at pH 8.0 was used to solubilize the epitopes of OW3 from theoocyst cell wall at 95° C. for 60 minutes. Following treatment, sampleswere microfuged at 21,000×g for 15 minutes at 4° C. A 450 μl aliquot ofsolubilized antigen supernatant was removed pending detection usingelectrochemiluminescence (ECL).

EXAMPLE 2 Preparation of Magnetic Capture Matrix

Monoclonal antibodies including anti-OW3 antibodies and other antibodiesspecific for C. parvum oocyst wall antigens unless otherwise specified,were provided by Dr. M. J. Arrowood (CDC, Atlanta, Ga.). IgM and IgGmonoclonal antibodies purified from mouse myeloma were purchased fromCalbiochem-Novabiochem (La Jolla, Calif.). Also used for antigendetection were 4.5 μm M450 rat anti-mouse IgM and M450 Rat anti-mouseIgG coated magnetic beads from Dynal, Inc. (Lake Success, N.Y.).Chromatography columns including Superose-6™ media, MONO-Q™, andFAST-Desalting™ columns were purchased from Pharmacia, Biotech, Inc.(Piscataway, N.J.).

For the C. parvum oocyst specific assay, M450 Rat anti-mouse IgM or IgGmagnetic beads were incubated at a concentration of 10⁷ beads/ml (0.075mg/ml) with 0.001 mg/ml 45% ammonium sulfate-precipitated,chromatography-purified monoclonal antibodies specific for particularepitopes of oocyst wall antigens. For OW3 monoclonal antibodies,anti-mouse IgM magnetic beads were used in the preparation. Incubationwas performed in disposable 12×75 mm borosilicate glass culture tubesand placed on an orbital shaker (approximately 800 rpm) for 60 minutesat room temperature. The beads were concentrated by placing the solutiononto a magnetic rack (MPC-6 magnetic rack, Dynal, Inc., Lake Success,N.Y.) for two minutes following which the solution containing unboundmonoclonal antibody was removed from the beads. Tubes were taken awayfrom the magnet and beads were resuspended gently in 2 ml ofelectrochemiluminescent diluent (ECL Diluent: 0.05M Tris-HCl, 0.5M NaCl,1% bovine serum albumin (BSA), 0.7% Tween-20, pH 8.0). Tubes were placedback on the magnetic rack, and the solution was removed as before. Thiswash procedure was repeated one more time. Washed beads were thenresuspended in ECL diluent to a final concentration of 2.5×10⁶ beads/ml(0.187 mg/ml) to yield 2.5×10⁵ beads per ECL assay (0.0187 mg/100 μl/ECLassay).

EXAMPLE 3 Preparation of Ruthenium Labeled Antibodies

Chromatography purified monoclonal antibodies as described in Example 2were conjugated to a ruthenium metal chelate TAG-NHS (an activatedruthenium label used for electrochemiluminescent detection) ester,referred to herein as the activated ruthenium label. The TAG-NHS esterused to label the detecting antibody was obtained from Igen, Inc.(Gaithersburg, Md.).

TAG-NHS was dissolved in dimethylsulfoxide (DMSO) for 15 minutes at aconcentration of 1.42×10⁻⁴ mM. Purified monoclonal antibodies in 0.01MNaPO₄ were combined with dissolved TAG at a challenge ratio of 15 to 30moles of TAG to one mole of monoclonal antibody (no amino groups werepresent during reaction). For coupling ruthenium to the OW3 monoclonalantibody, a challenge ratio of 30 to 1 was used in the reaction toachieve an approximately 18 to 1 final incorporation ratio. The mixturewas incubated for 60 minutes at room temperature, in the dark, on anorbital shaker. After coupling, Tris-HCl was added to a finalconcentration of 0.2M to stop the reaction. Unbound TAG was removed bydesalting using a FAST-Desalting™ column (Pharmacia, Biotech, Inc.,Piscataway, N.J.) into 0.05M Tris-HCl, 0.5M NaCl, pH 8.0. A finalruthenium to immunoglobin molar incorporation ratio for each monoclonalantibody was determined by measuring the protein concentration of theconjugate and its absorbance at 455 nm. This ratio was calculatedaccording to reagent protocol supplied by Igen, Inc. (Gaithersburg,Md.). A 3X concentration of MAb-Ru²⁺ conjugate was prepared in ECLdiluent at 3×10⁻⁴ mg/ml protein and was used in the assay at a finalconcentration of 10⁻⁴ mg/ml.

EXAMPLE 4 Electrochemiluminescent Immunoassay

Duplicate tests of each sample were performed for both C. parvumoocyst-specific and non-specific tests. A 100 μl aliquot of solubilizedantigen supernatant, prepared as described in Example 1 above, wasincubated with 100 μl of M450 Rat anti-mouse monoclonal antibodymagnetic beads, prepared as described in Example 2 above, and 100 μl ofRu²⁺-labeled monoclonal antibodies, prepared as described in Example 3above, on an orbital shaker (˜800 rpm) for 60 minutes at roomtemperature. Following incubation, samples were placed on the carouselof an ORIGEN™ ANALYZER (Igen, Inc., Gaithersburg, Md.) and assayedaccording to instrument specifications.

EXAMPLE 5 Determination of Number of Oocysts in Test Samples

A determination of the number of C. parvum oocysts detected in the testsamples, as described in Example 4 above, first requires subtraction ofthe ECL counts of the negative sample from test samples and positivecontrols. ECL counts of the positive controls and test samples obtainedfrom the non-specific assay are subtracted from the ECL counts ofcorresponding samples obtained from the specific assay. A standard curveis constructed using the net ECL counts obtained from the positivesamples, to be used in the calculation of the number of oocysts in thetest samples. For a 1 ml sample volume, ten times the calculated amountof oocyst will be present in the sample.

The following formula was used to calculate the total number of oocystsin 100 liters of water sample;

T=[(10N×E)/(Q/100)]×(100%/R)

T=total number of oocysts in 100 L of water sample

N=number of oocysts/ 0.1 mL in the test sample (obtained from standardcurve)

E=the total volume of the filtrate sample (mL)

Q=the total volume of the water sample collected through cartridgefilter (L)

R=theoretical oocyst recovery percentage of filter cartridge used

Results of Electrochemiluminescent Assay on Test Samples Containing C.parvum Oocysts

Different numbers of oocysts (10¹ to 10⁶) were spiked into the buffer orsediment samples having various nephelometric turbidity unit (NTU)values (5077 NTU, 4125 NTU and 330,000 NTU), then assayed for thepresence of soluble OW3 antigen by electrochemiluminescence as describedabove. As shown in FIGS. 1 and 2, an increase in turbidity did notadversely affect the sensitivity or accuracy of the assay. As also shownin FIGS. 1 and 2, the assay is capable of detecting as few as 1000oocysts per milliliter sample.

In FIGS. 3 and 4, 10³ to 10⁵ oocysts from various Cryptosporidiumspecies were spiked into fecal suspension samples and assayed for thepresence of solubilized OW3 antigen. The graph in FIG. 3 shows thespecificity of the current method for C. parvum. FIG. 4 compares testresults for C. parvum and C. baileyi and shows that the assay, utilizinga monoclonal antibody specific for C. parvum, fails to cross-react withC. baileyi.

Modifications and variations of the present method will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe appended claims.

We claim:
 1. A method for detecting Cryptosporidium in a samplecomprising: (a) incubating the sample with a buffered solutioncomprising a synthetic zwitterionic compound, wherein the incubation isat a temperature of greater than 75° C. for a sufficient amount of timeto result in a protein or a glycoprotein antigen of a Cryptosporidiumoocyst being solubilized in the buffered solution, (b) reacting theantigen of the Cryptosporidium oocyst in the buffered solution with anantibody that specifically binds the solubilized antigen to form anantibody-antigent complex; and (c) detecting the antibody-antigencomplex in the buffered solution by an immunoassay method, wherein thedetection of the antigen-antibody complex indicates the presence ofCryptosporidium in the sample.
 2. The method of claim 1, wherein theprotein or glycoprotein is a membrane-bound protein or glycoprotein ofan oocyst wall of the Cryptosporidium.
 3. The method of claim 1, whereinthe sample contains less than 50,000 oocysts/ml.
 4. The method of claim1, wherein the Cryptosporidium is Cryptosporidium parvum.
 5. The methodof claim 1, wherein the antibody is bound to a solid phase.
 6. Themethod of claim 1, wherein the detection is by electrochemiluminescence.7. The method of claim 1, further comprising incubating theantibody-antigen complex with a second antibody specifically binds theantigen or the antibody-antigen complex, wherein the second antibody islabeled with a detectable label.
 8. The method of claim 7, wherein thelabel is selected from the group consisting of anelectrochemiluminescent label, a chemiluminescent label, an enzymaticlabel, a bioluminescent label, and a fluorescent label.
 9. The method ofclaim 8, wherein the electrochemiluminescent label is selected from thegroup consisting of ruthenium and aequorin.
 10. The method of claim 1,wherein the antigen is an epitope of the OW3 antigen of theCryptosporidium parvum oocyst.
 11. The method of claim 1, wherein thesample is incubated with the buffered solution comprising the compoundfor at least 30 minutes.
 12. The method of claim 1, wherein the sampleis a water sample.
 13. The method of claim 12, wherein the water sampleis selected from the group consisting of natural bodies of water,community water reserviors, and recreational waters, wherein therecreational waters are selected from the group consisting of swimmingpools, whirlpools, hot tubs, spas, water parks, naturally occurringfresh waters, and marine surface waters.
 14. The method of claim 1,wherein the sample is a biological sample.
 15. The method of claim 14,wherein the sample is a fecal sample.
 16. An assay kit for the detectionof Cryptosporidium in a sample comprising, (a) an antibody specific fora solubilized Cryptosporidium oocyst antigen wherein the antigen is aprotein or glycoprotein, and (b) a buffered solution comprising acompound selected from the group consisting ofn-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-Decyl-N,N-dimethyl-3-ammonio-1propanesulfonate,n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, wherein thebuffered solution solubilizes the Cryptosporidium oocyst antigen presentin the sample.
 17. The assay kit of claim 16, wherein the antibody isimmobilized on a solid phase.
 18. The assay kit of claim 16, furthercomprising a labeled antibody that specifically binds the solubilizedantigen.
 19. The method of claim 1, wherein the sample is a turbid watersample.
 20. The assay kit of claim 16, wherein the sample is a turbidwater sample.
 21. The method of claim 1, wherein the sample containsless than 10,000 oocysts/ml.
 22. The method of claim 1, wherein thesample contains less than 1,000 oocysts/ml.
 23. The method of claim 1,wherein the antibody is a monoclonal antibody.
 24. The method of claim1, wherein the sample has a turbidity of up to 300,000 NTU.
 25. Themethod of claim 1, wherein the buffered solution has a pH of less thanpH=7 and greater than pH=4.
 26. The method of claim 1, wherein thebuffered solution has a pH of greater than pH=7 and less than pH=10. 27.The method of claim 1, wherein the compound is selected from the groupconsisting ofn-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonateand n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.
 28. Themethod of claim 1, wherein the detecting is not by microscopy.